In vitro-expanded PBMC were incubated in medium www.selleckchem.com/products/Axitinib.html alone (control) or with viral peptides (5 ��g/ml) for 5 h in the presence of brefeldin A (10 ��g/ml). After a washing, the cells were stained with anti-CD8 PE-Cy7 and anti-CD3 peridinin chlorophyll protein-Cy5.5 monoclonal antibody (MAb) for 30 min at 4��C and then fixed and permeabilized using Cytofix/Cytoperm fixation/permeabilization solution (BD Biosciences, San Jose, CA), according to the manufacturer’s instructions. The cells were stained with anti-IFN-�� PE for 30 min on ice, washed, and analyzed by flow cytometry. To assess degranulation activity, CD107a PE antibody (BD Pharmingen, San Diego, CA) was added to all wells at the beginning of the 5-h incubation with T cells. Following the incubation, the cells were washed and labeled with anti-CD8 PE-Cy7.

CTL clones and EBV-transformed B-cell lines. HBV Core18-27-specific CD8+ T-cell clones were generated from HLA-A2+ HBV patients with acute hepatitis B as previously described (17). Epstein-Barr virus (EBV) B-cell lines with known HLA-A2 subtypes (kindly provided by Chan Soh Ha, Department of Microbiology, National University of Singapore) were grown and maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 20 mM HEPES, 0.5 mM sodium pyruvate, 100 U/ml penicillin, 100 ��g/ml streptomycin, MeM amino acids with l-glutamine, MeM nonessential amino acids (Invitrogen, Carlsbad, CA), and 5 ��g/ml Plasmocin (InvivoGen, San Diego, CA) to prevent mycoplasma contamination. IFN-�� ELISPOT assay.

IFN-�� enzyme-linked immunospot (ELISPOT) assays were performed as previously described (7) using a panel of 313 overlapping peptides covering the entire peptide sequence of HBVgenB or HBVgenD pooled in the described mixtures and used in patients infected with the respective HBV genotype. HBV-specific T-cell responses were analyzed in IFN-�� ELISPOT assays either ex vivo using fresh or frozen PBMC or after short-term peptide-specific polyclonal T-cell expansion (10 days). Briefly, 96-well plates (Multiscreen-HTS; Millipore, Billerica, MA) were coated overnight at 4��C as recommended by the manufacturer with 5 ��g/ml capture mouse anti-human IFN-�� MAb (1DIK; Mabtech, Sweden). The plates were then washed five times with phosphate-buffered saline and blocked with AIM-V supplemented with 10% heat-inactivated fetal calf serum for 30 min at room temperature. A total of 1 �� 105 PBMC or 5 �� 104 cells from short-term polyclonal T-cell lines were seeded per well, in duplicate for each individual peptide mixture. The plates were incubated for 18 h at 37��C in the presence or absence of peptides (at a final concentration of 5 ��g/ml). After five washes with phosphate-buffered saline, Entinostat 100 ��l of 0.